The drug discovery process is currently undergoing a fundamental revolution as it embraces “functional genomics,” that is, high throughput genome- or gene-based biology. This approach is rapidly superceding earlier approaches based on positional cloning. A phenotype, that is a biological function or genetic disease, would be identified and this would then be tracked back to the responsible gene, based on its genetic map position.
Functional genomics relies heavily on the various tools of bioinformatics to identify gene sequences of potential interest from the many molecular biology databases now available. There is a continuing need to identify and characterize further genes and their related polypeptides/proteins, as targets for drug discovery with the potential for affecting immune response.
The strategy of innate immune recognition is based on the detection of constitutive and conserved products of microbial metabolism. Many metabolic pathways and individual gene products are unique to microorganisms and absent from host cells. Although these targets of recognition are not absolutely identical between different species of microbes, the gene products may be found in the context of a common molecular pattern, which is typically highly conserved and invariant among microbes of a given class. Because the targets of innate immune recognition are conserved molecular patterns, they are called pathogen associated molecular patterns (PAMPs).
The recent discovery and characterization of the Toll-like receptor (TLR) family have incited new interest in the field of innate immunity. TLRs are pattern recognition receptors that have a unique and critical function in animal immunity. TLRs typically are transmembrane receptors characterized by an extracellular leucine rich repeats domain and an intracellular TIR (Toll/Interleukin-1 Receptor) domain. The TIR domain is a conserved protein-protein interaction module and plays a role in host defense. In other words, TLRs play a critical role in microbial recognition and control of adaptive immune responses.
In mammalian species, there are at least ten (10) TLRs, and each has a distinct function in innate immune recognition. The TLRs mainly differ from one another with regard to ligand specificity, the use of accessory molecules, expression profiles and differences in signal transduction pathways.
Human TLR4 was the first identified and functionally characterized mammalian Toll. TLR4 functions as the signal transducing receptor for the PAMP lipopolysaccharide (LPS) as well as other PAMPs, which are apparent to one skilled in the art.
Activation of signal transduction pathways by TLRs leads to the induction of a variety of genes that function in host defense including inflammatory cytokines, chemokines, MHC and co-stimulatory molecules. Mammalian TLRs also induce multiple effector molecules such as inducible nitric oxide synthetase and antimicrobial peptides that can directly destroy microbial pathogens.
The signaling pathway, which appears to be shared by all members of the Toll and Interleukin-1 Receptor (IL-1R) families, includes four essential components: the adapters TRAF6, MyD88 and Tollip and a protein kinase, IRAK. MyD88 contains two protein interaction domains: an N-terminal death domain and a C-terminal TIR domain. The TIR domain of MyD88 associates with the TIR domain of TLR and IL-1R, while the death domain interacts with the death domain of IRAK.
In cells wherein MyD88 expression has been suppressed (i.e. in MyD88 knockout mice), macrophages and dendritic cells do not produce cytokines IL-1β, TNF-α, IL-6 and IL-12 when stimulated with LPS, MALP-2 or CpG, which signal through TLR4, TLR2 and TLR9, respectively. Consequently, MyD88 knockout mice are resistant to endotoxic shock. Furthermore, when normal bone marrow-derived dendritic cells (BMDCs) are stimulated with LPS or CpG, they produce large amounts of IL-12 and upregulate cell surface expression of MHC and co-stimulatory molecules. However, in MyD88 deficient BMDCs, stimulation with LPS or CpG does not produce IL-12 or IL-6.
Additionally, RNA-dependent protein kinase (PKR)-deficient cells fail to activate c-Jun N-terminal Kinase (JNK) and p38 MAP Kinase (p38) in response to LPS stimulation. As TLR4 is required for signals downstream of LPS, this indicates that PKR is a component of the TLR4 signaling pathway. Although phosphorylated PKR can be detected in LPS stimulated wild-type macrophages, phosphorylated PKR has also been detected in LPS stimulated MyD88-deficient macrophages. Interestingly, PKR from the MyD88-deficient macrophages was activated with slower kinetics.
Although some cellular responses are completely abolished in MyD88-deficient cells, TLR4, but not TLR9 or TLR2, can still activate NF-κB and MAP kinases. This difference indicates that another adapter protein exists that can mediate MyD88-independent signaling in response to TLR4 ligation.
Thus, there is a need to determine the structure and function of the adapter protein involved in the MyD88 independent signaling downstream of TLR4 for various purposes including to develop compounds to treat diseases related to TLR4 function.